Composition comprising lactobacillus plantarum cjlp475 strain and lactobacillus plantarum cjlp17 strain and use thereof

ABSTRACT

The present disclosure relates to a composition comprising a  Lactobacillus plantarum  CJLP475 strain deposited under Accession No. KCCM12287P; and a  Lactobacillus plantarum  CJLPl7 strain deposited under Accession No. KCCM12249P, and use thereof.

TECHNICAL FIELD

The present disclosure relates to a composition including aLactobacillus plantarum CJLP475 strain and a Lactobacillus plantarumCJLP17 strain, which have acid-resistance, bile-resistance, andantiviral and immune-enhancing activities.

BACKGROUND ART

In the current livestock industry, livestock are killed due to highlycontagious viral diseases, and this often leads to economic damage tofarms. In particular, in the swine industry, infectious diseases causedby viruses and germs, such as porcine respiratory disease complex,postweaning multisystemic wasting syndrome, porcine reproductive andrespiratory syndrome, porcine epidemic diarrhea, which are the fourmajor chronic wasting diseases, have caused huge economic losses.

Among them, porcine epidemic diarrhea is a porcine digestive diseasecaused by the infection of porcine epidemic diarrhea virus (PEDV), amember of the corona virus family. The virus proliferates in the villiof the small intestine and large intestine and causes acute enteritis,vomiting, and watery diarrhea in pigs of all ages, especially inpiglets. In particular, the damage is severe mainly in winter, fromNovember to April, and it is known that the mortality rate ofpre-weaning piglets within 1 week of birth is about 50%, and in severecases, the mortality rate can reach almost 100% due to extremedehydration.

The PED virus was first recognized in Europe in 1971, and G1a type PEDVCV777 was further identified and separated in Belgium in 1976. The virushad spread through Europe in 1980s, and the outbreaks have occurred inEast Asian countries including China, Korea, Japan, Taiwan in 1990s.Further, G2b type PEDV, which is more virulent than the G1a type, hadfirst emerged in China in 2010. The new type PEDV has spread to NorthAmerica (the United State and Canada) and further to Southeast Asia andEurope, causing severe damage. In 2013, the damage was estimated toabout 2.2 trillion won due to the loss of productivity in the US swineindustry. In Korea, it is reported that an outbreak of PEDV annuallyoccurs in 20 to 40% of pig farms, causing 6% of the total pigs to bekilled. It is also reported that the infection rate of the vehiclesentering and leaving slaughterhouses reaches about 60% (Korea RuralEconomic Institute, Korea Swine Veterinary Association).

Until now, the only way to prevent the G2b type PED virus is by thoroughsterilization. Many farms use an artificial infection method or theexisting G1a type PEDV vaccine to prevent the damage caused by viraldiseases, but there is a limitation in preventing the G2b type PEDVinfection. In addition, the newly developed inactivated G2b type PEDVvaccine is also being used. However, several problems have been raisedin regard to the preventive measures of PEDV, such as showing alimitation of the inactivated vaccine for the prevention of digestivediseases because the inactivated vaccines are only used instead of thedevelopment of live vaccines. In order to overcome such problems,development of agents for prevention and treatment of PED virus(vaccines etc.) and treatment (IgY, essential oil, organic acid,probiotics, etc.) is actively carried out. In particular, a method ofenhancing immunity using a functional material that stimulates theimmune system in vivo while having an antiviral effect has been recentlystudied.

Immunity is generally divided into innate immunity and adaptiveimmunity. Innate immunity is a system that instantly defends pathogeninfection from the first line, acting directly on invaders (antigens) orinducing adaptive immunity. Adaptive immunity is a more complex andprecise system that recognizes and removes invaders, or acts as a memoryfor the corresponding invaders, thereby providing more permanent immunefunctions compared to the innate immunity. Dendritic cells (DCs),macrophages, and natural killer cells, which are antigen-presentingcells related to innate immunity, directly serve innate immune functionsand possess receptors that assist in activation of various types ofT-cells, thereby secreting cytokines. Adaptive immunity is a secondarydefense system against antigens that have entered the body, and is aspecific immune response carried out by B lymphocytes and T lymphocytes.The immune responses controlled by antigen-activated T cells include acytotoxic T cell response and a helper T cell response. The dendriticcells, macrophages, and natural killer cells related to the innateimmunity also recognize foreign invasive substances and secrete variouskinds of cytokines such as IL-12 and IL-4 to thereby induce a responsethat appropriately changes the immunity of the host animal, and thus mayprovide the immune defense mechanism in a suitable direction. Naive CD4T cells, which act as precursors of T cells, are differentiated by keycytokines. For example, if IL-12 (interleukin-12) is present in a highconcentration, CD4 T cells promote the differentiation of Th1 (Thelper 1) cells, thereby inducing a CTL (cytotoxic T lymphocyte)response that eradicates intracellular pathogens, whereas, if IL-4(interleukin-4) is present in a high concentration, they induce aresponse that specifically eradicates extracellular pathogens (antibodysecretion of B cells). In addition, the above-enumerated immune cellsrespond in an appropriate manner to invaders that have entered the bodythrough an elaborate and complex process, by secreting TGF-beta(Transforming growth factor Beta) and IL-10 to suppress excessive immuneresponses such as an inflammatory reaction, secreting TGF-beta toinhibit excessive immune response and thereby activates regulatory Tcells, stimulating the production of antibodies by transforming B cellsinto plasma cells in response to the secretion of TGF-beta and IL-6(interleukin-6), or inducing an immune response (Th17) to eradicatefalse autoimmunity and extracellular pathogens. However, such immuneresponses sometimes require an additional and appropriate immuneenhancer due to imbalanced or poor immune responses.

DISCLOSURE Technical Problem

The present inventors have completed the present disclosure by isolatingand identifying microorganisms capable of activating the immune systemwhile exhibiting an inhibitory activity against the above-mentionedvirus, and confirming their activities.

Technical Solution

It is one object of the present disclosure to provide a compositionincluding:

(a) a Lactobacillus plantarum CJLP475 strain deposited under AccessionNo. KCCM12287P; and

(b) a Lactobacillus plantarum CJLP17 strain deposited under AccessionNo. KCCM12249P.

According to one embodiment of the present disclosure, the (a) and (b)may be in the form of a strain itself, a lysate thereof, a culturethereof, a concentrate thereof or a dried form thereof.

It is another object of the present disclosure to provide a feed or afeed additive including the aforementioned composition.

It is still another object of the present disclosure to provide a foodincluding the aforementioned composition.

It is still further another object of the present disclosure to providea cosmetic including the aforementioned composition.

It is still further another object of the present disclosure to providea pharmaceutical including the aforementioned composition.

It is still further another object of the present disclosure to providea method for enhancing immunity of a subject, including administeringthe aforementioned composition to a subject in need.

It is still further another object of the present disclosure to providea method for preventing or treating a virus-infected disease of asubject, including administering the aforementioned composition to asubject in need.

It is still further another object of the present disclosure to providethe use of the composition for prevention or treatment of virus-infecteddisease.

Advantageous Effects

The composition of the present disclosure has high acid-resistance andbile-resistance and can thus be provided as a probiotic. The compositionactivates immune cells in vivo and thereby regulates immune functions,and particularly exhibits an excellent inhibitory activity againstPorcine epidemic diarrhea virus (PEDV) infection. In addition, it can befound that when the composition of the present disclosure is orallyadministered to sows, and the piglets that have been delivered from thesows are challenged with PED virus, the mortality of the piglets can beremarkably reduced. Accordingly, the present disclosure can provide acomposition having an antiviral activity against PED virus, animmunity-enhancing activity, and an effect of improving weight gain andreducing diarrhea incidence in livestock, and thus, the composition canbe effectively used as a feed composition or a composition for feedadditives, a food composition, a cosmetic composition or apharmaceutical composition.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows images of blood agar plate confirming no hemolytic activityof the Lactobacillus plantarum CJLP475 strain and the Lactobacillusplantarum CJLP17 strain.

FIG. 2 is a graph showing no cytotoxicity of the composition includingthe Lactobacillus plantarum CJLP475 strain and the Lactobacillusplantarum CJLP17 strain.

FIG. 3 shows microscopic images illustrating the inhibitory effectagainst PED virus infection by the composition including theLactobacillus plantarum CJLP475 strain and the Lactobacillus plantarumCJLP17 strain.

FIG. 4 is a graph showing the rate of increase of vaccine-specific IgGantibodies in the serum of the sows fed with the composition includingthe Lactobacillus plantarum CJLP475 strain and the Lactobacillusplantarum CJLP17 strain.

FIG. 5 is a graph showing the rate of increase of vaccine-specific IgAantibodies in the serum of the sows fed with the composition includingthe Lactobacillus plantarum CJLP475 strain and the Lactobacillusplantarum CJLP17 strain.

FIG. 6 is a graph showing the neutralizing antibody titer in colostrumof the sows fed with the composition including the Lactobacillusplantarum CJLP475 strain and the Lactobacillus plantarum CJLP17 strain.

FIG. 7 is a graph showing the content of vaccine-specific IgG antibodiesin piglet serum after a PED virus challenge in piglets born from thesows fed with the composition including the Lactobacillus plantarumCJLP475 strain and the Lactobacillus plantarum CJLP17 strain.

FIG. 8 is a graph showing the content of TGF-beta in piglet serum aftera PED virus challenge in piglets born from the sows fed with thecomposition including the Lactobacillus plantarum CJLP475 strain and theLactobacillus plantarum CJLP17 strain.

FIG. 9 is a graph showing the V/C ratio of piglet jejunum after a PEDvirus challenge in piglets born from the sows fed with the compositionincluding the Lactobacillus plantarum CJLP475 strain and theLactobacillus plantarum CJLP17 strain.

FIG. 10 is a graph showing a goblet cell density of piglet colon after aPED virus challenge in piglets born from the sows fed with thecomposition including the Lactobacillus plantarum CJLP475 strain and theLactobacillus plantarum CJLP17 strain.

FIG. 11 is a graph showing the mean body temperature for 7 days after aPED virus challenge in piglets born from the sows fed with thecomposition including the Lactobacillus plantarum CJLP475 strain and theLactobacillus plantarum CJLP17 strain.

FIG. 12 is a graph showing the diarrhea incidence for 7 days after a PEDvirus challenge in piglets born from the sows fed with the compositionincluding the Lactobacillus plantarum CJLP475 strain and theLactobacillus plantarum CJLP17 strain.

FIG. 13 is a graph showing the survival rate for 7 days after a PEDvirus challenge in piglets born from the sows fed with the compositionincluding the Lactobacillus plantarum CJLP475 strain and theLactobacillus plantarum CJLP17 strain.

DETAILED DESCRIPTION OF EMBODIMENTS

Hereinafter, the present disclosure will be described in more detail.Meanwhile, each description and embodiment disclosed herein can beapplied to other descriptions and embodiments, respectively. That is,all combinations of various elements disclosed herein fall within thescope of the present disclosure. Further, the scope of the presentdisclosure is not limited by the specific description described below.

One aspect of the present disclosure to overcome the objects above,there is provided a composition including:

(a) a Lactobacillus plantarum CJLP475 strain deposited under AccessionNo. KCCM12287P; and

(b) a Lactobacillus plantarum CJLP17 strain deposited under AccessionNo. KCCM12249P.

Specifically, the composition may have an antiviral, acid-resistance,bile-resistance, and an immune-enhancing activity.

As used herein, the term “Lactobacillus” is a microorganism of aerobicor facultative anaerobic gram-positive bacillus widely distributed innature. The microorganisms belonging to the genus Lactobacillus includeLactobacillus plantarum, etc. The present inventors provide novelstrains belonging to the Lactobacillus plantarum, which were depositedunder Accession Nos. KCCM12287P and KCCM12249P, and are designated as“Lactobacillus plantarum CJLP475” and “Lactobacillus plantarum CJLP17”,respectively. They correspond to a probiotic strain, are harmless to thehuman body and can be used without side effects.

As used herein, the term “probiotics” refers to live bacteria that enterthe body and provide a healthy benefit. Most of the probiotics known sofar have been consumed through fermented milk products made from lacticacid bacteria such as Lactobacillus. In recent years, however,probiotics are available on the market in the form of fermented milk,granules, powder and the like, containing some of the bacteria such asBifidobacterium and Enterococcus, in addition to Lactobacillus. TheLactobacillus plantarum CJLP475 and the Lactobacillus plantarum CJLP17of the present disclosure may also be used in the form of fermentedmilk, granules, powder and the like, but are not limited thereto.

As used herein, the term “acid-resistance” refers to the property ofwithstanding high acidity. If the probiotics are acid-resistant, theycan be prevented from being degraded or damaged even when exposed tostrong acidic conditions in the stomach, by consumption through variousroutes of administration including oral administration.

As used herein, the term “bile-resistance” refers to the resistance todigestive enzymes in the bile. The bile is made from the liver andstored in the gallbladder, and is a weak alkaline greenish brown liquidthat helps the digestion of fat in the duodenum of the small intestines.It emulsifies fat to help digestion and absorption. The bile is one ofthe major causes of reducing the effect of probiotic administration asthey act on probiotics ingested through various routes including oraladministration and the like.

Specifically, among the compositions of the present disclosure, theLactobacillus plantarum CJLP475 strain was isolated from soy sauce(i.e., a traditional fermented food), and the Lactobacillus plantarumCJLP17 strain was isolated from the feces and colostrum of sows andpiglets in domestic farms where the PED virus had occurred. Themorphological characteristics of the strains is that each strain is agram-positive bacillus, and that the Lactobacillus plantarum CJLP475strain is represented by 16s rDNA nucleotide sequence of SEQ ID NO: 1,and the Lactobacillus plantarum CJLP17 strain is represented by 16s rDNAnucleotide sequence of SEQ ID NO: 2. Each of the nucleotide sequenceswas analyzed and found to be about 99% homologous with Lactobacillusplantarum. Accordingly, the present inventors deposited the novelisolated Lactobacillus plantarum CJLP17 strain at the Korean CultureCenter of Microorganisms, an International Depositary Authority, underBudapest Treaty on Apr. 13, 2018, with Accession No. KCCM12249P and theLactobacillus plantarum CJLP475 strain at the Korean Culture Center ofMicroorganisms, an International Depositary Authority, under BudapestTreaty on Jul. 11, 2018, with Accession No. KCCM12287P.

In order to stably maintain the Lactobacillus plantarum CJLP475 strainand the Lactobacillus plantarum CJLP17 strain of the present disclosurefor a long period of time, the strains may be stored by dissolving thecells in a storage solution prepared by mixing a certain amount ofglycerol in water at −70° C., or may be freeze-dried by suspending thecells in sterilized 10% skim milk, but are not limited thereto, and maybe stored for a long time by various known methods.

The composition of the present disclosure may not exhibit a hemolyticactivity against red blood cells. Hemolysis refers to the destruction ofred blood cells and the release of hemoglobin to the surrounding area,and is an action by which the red blood cells are hemolyzed by enzymesproduced from harmful bacteria in vivo. Therefore, even if thecomposition is administered in vivo, it may not cause hemolysis in theblood vessel.

In addition, the composition of the present disclosure may have a weakresistance or no resistance to antibiotics. The antibiotics mayspecifically be, but are not limited to, ampicillin, clindamycin,gentamicin, kanamycin, erythromycin, ampicillin/sulbactam,chloramphenicol, or streptomycin. Accordingly, even when the compositionis used in pharmaceuticals, health functional foods, feed additives, orthe like, it has no resistance to antibiotics, and thus, the possibilityof causing relevant pharmacological effects or environmental problems islow.

The composition may enhance the activation of immune cells to increasesecretion of cytokines, or may be administered in vivo to promote immunefunction.

As used herein, the term “immune cells” refer to all cells that play arole in immune function in vivo, and can be largely divided into T cellsand B cells. The immune cells may include, but are not limited to, Th1or Th2 cells. The composition of the present disclosure may have anactivity to stimulate immune cells and thereby increase the secretion ofcytokines such as IL-12, IL-10, or TGF-beta.

In viral diseases, which generally show a high mortality rate, necrosisof cells or tissues themselves due to the virus can lead to a secondaryinfection and septicemia induced by other bacteria, an inflammatorydisease caused by an over-activated immune response, or appetitereduction and dehydration. Therefore, when the antiviral efficacy(relevant to Th1, Th2), which suppresses the viral infection, and theimmune response (Th2, anti-inflammation), which regulates the secondaryinfection and the excessive inflammation reaction, are simultaneouslyenhanced, an effective prevention and treatment effect for viraldiseases can be achieved. As such, a method for simultaneously enhancingTh1 and Th2 in regards to providing an immune-enhancing effect throughprobiotics is not known in the art, and such a method has been newlydiscovered by the present inventors. In addition, the composition of thepresent disclosure has an immunomodulatory ability to regulate theTh1/Th2 imbalance.

As used herein, the term “cytokine” refers to a glycoprotein used as asignal substance for controlling and stimulating a body defense system,and may be, for example, IL-12, IL-10, or TGF-beta, but is not limitedthereto.

The composition may promote the growth of a subject or reduce diarrheaincidence upon administration to a subject.

As used herein, the term “subject” may refer to all animals includinghumans in which the immunity is weakened or likely to be weakened. Forexample, the subject may include animals excluding humans or includinghumans. The animal may include not only humans, but also all animalsthat need the above-mentioned efficacy to be revealed, and mayspecifically be mammals such as cows, horses, sheep, pigs, goats,camels, antelopes, dogs, cats, and the like, or alternatively, it may bea livestock or pet.

The above administration method is not particularly limited, but may beadministered through various routes including an oral or parenteralroute as long as it can reach the target tissues. Examples thereof maybe an oral administration.

The composition may increase antibodies in the body. The immunity of asubject can be improved by increasing the secretion of the antibodiesinvolved in the immune function. The composition may be administered toa subject, and the administration method is not particularly limited,but may be administered through various routes including an oral orparenteral route as long as it can reach the target tissues. Examplesthereof may be an oral administration. The body may be a body fluid suchas blood or colostrum, but is not limited thereto. The antibody may bean IgG, an IgA, or a neutralizing antibody, but is not limited thereto.

The composition, when administered to a subject, may enhance theimmunity in the offspring born from the subject through the maternalantibody. The enhancement of the immunity may include an increase in theantibody or cytokine secretion, but is not limited thereto.

The composition, when administered to a subject, may alleviate thesymptoms of viral infection in the offspring born from the subject. Thevirus may be porcine epidemic diarrhea virus (PEDV), and may includewithout limitation any virus, whose infection can be inhibited by thecomposition of the present disclosure. The alleviation of the symptomsmay include, but is not limited to, alleviating the conditions ofdigestive organs, maintaining normal body temperature, reducingdiarrhea, and increasing survival rate.

The composition may have an antiviral activity against the porcineepidemic diarrhea virus (PEDV).

As used herein, the term “anti-virus” refers to the property ofinhibiting a viral infection. Probiotics cannot inactivate a virusthemselves, but can increase the immunity of a virus-infected subject byan immunological activity, thereby allowing to resist the viralinfection.

Porcine epidemic diarrhea virus is coronavirus that infects the cellslining of the small intestine of pigs, causing porcine epidemic diarrheawhich induces severe diarrhea and dehydration. The virus may include anyvirus, whose infection can be inhibited by the composition of thepresent disclosure, without limitation.

The strains corresponding to (a) and (b), respectively, in thecomposition of the present disclosure may be in the form of a strainitself, a lysate thereof, a culture thereof, a concentrate thereof or adried form thereof. When the strains are treated on a subject, theactivity and infection of PED virus can be remarkably suppressed. Thus,the composition may be used as an antiviral composition against PEDvirus, or a pharmaceutical, a functional food composition, a quasi-drugcomposition or a feed composition for preventing or improving porcineepidemic diarrhea.

According to one embodiment of the present disclosure, the (a) and (b)in the composition of the present disclosure may be in the form of astrain itself, a lysate thereof, a culture thereof, a concentratethereof, or a dried form thereof, but are not limited thereto. Furtherdetails regarding the composition can be made reference to the abovedescription.

The strains of the present disclosure can be cultured by a conventionalmethod for culturing Lactobacillus strains. As the medium, a naturalmedium or a synthetic medium can be used. As the carbon source of themedium, for example, glucose, sucrose, dextrin, glycerol, starch and thelike may be used. As the nitrogen source, peptone, meat extracts, yeastextracts, dried yeasts, soybean, ammonium salts, nitrate, and otherorganic or inorganic nitrogen-containing compounds may be used, but isnot limited thereto. As the inorganic salts included in the medium,magnesium, manganese, calcium, iron, potassium, and the like may beused, but is not limited thereto. Amino acids, vitamins, nucleic acidsand related compounds may be added to the medium in addition to thecarbon source, the nitrogen source and the components of the inorganicsalt. The composition of the present disclosure may be cultured for 12hours to 4 days in a temperature range of 20° C. to 40° C.

In the present disclosure, the culture broth may be referred to thecomposition after the completion of culture, and more specifically, theculture broth may or may not include cells. Therefore, the culture brothmay include a culture supernatant, a composition from which a culturesupernatant is removed, or a concentrated composition thereof. Thecomposition of the culture broth may additionally contain not onlycomponents required for conventional culture of Lactobacillus, but alsocomponents that act synergistically to the growth of Lactobacillus, andthe compositions thereof may be readily selected by those skilled in theart.

In addition, the strain may be in a liquid state or a dry state, and thedrying method may include, but not limited to, air drying, naturaldrying, spray drying, and freeze drying.

The composition may be a complex preparation containing two types ofstrains, in which (a) a Lactobacillus plantarum CJLP475 strain depositedunder Accession No. KCCM12287P; and (b) a Lactobacillus plantarum CJLP17strain deposited under Accession No. KCCM12249P are contained at thesame time. Accordingly, the composition may be merely composed of twotypes of strains corresponding to (a) and (b), lysates thereof, culturesthereof, concentrates thereof, or dry products thereof, but is notlimited thereto. In this case, the two types of strains corresponding tothe above (a) and (b) may not necessarily be in the same form in thecomposition, and may be in a combination of forms, if necessary, the (a)may be in the form of a strain itself, and (b) may be in the form of alysate of the strain, but are not limited thereto.

In the composition, the concentration of the mixture of theLactobacillus plantarum CJLP475 strain and the Lactobacillus plantarumCJLP17 strain may be, but is not limited to, 10⁵ CFU/mL to 10¹⁰ CFU/mL,10⁵ CFU/mL to 10⁹ CFU/mL, 10⁵ CFU/mL to 10⁸ CFU/mL, 10⁵ CFU/mL to 10⁷CFU/mL, 10⁵ CFU/mL to 10⁶ CFU/mL, 10⁶ CFU/mL to 10¹⁰ CFU/mL, 10⁷ CFU/mLto 10¹⁰ CFU/mL, 10⁵ CFU/mL to 10¹⁰ CFU/mL, 10⁹ CFU/mL to 10¹⁰ CFU/mL,10⁶ CFU/mL to 10⁹ CFU/mL, 10⁶ CFU/mL to 10⁸ CFU/mL, 10⁶ CFU/mL to 10⁷CFU/mL, 10⁷ CFU/mL to 10⁹ CFU/mL, 10⁷ CFU/mL to 10⁸ CFU/mL, or 10⁸CFU/mL to 10⁹ CFU/mL.

The composition may further include a cryoprotectant or an excipient.The cryoprotectant or excipient may be a non-naturally occurringsubstance or a naturally occurring substance, but is not limitedthereto. In another embodiment, the cryoprotectant or excipient may be asubstance that does not naturally contact with the Lactobacillusplantarum CJLP475 strain and the Lactobacillus plantarum CJLP17 strain,or a substance that is not naturally contained simultaneously with thetwo strains, but is not limited thereto. In still another embodiment,the composition may further include at least one cryoprotectant selectedfrom the group consisting of glycerol, trehalose, maltodextrin, skimmilk powder and starch, and/or at least one excipient selected from thegroup consisting of glucose, dextrin and skim milk. The cryoprotectantof the present disclosure may be contained in an amount of 0.01% to 20%by weight and 0.01% to 10% by weight based on the total weight of thecomposition. Specifically, the glycerol may be contained in an amount of5% to 20% by weight, the trehalose may be contained in an amount of 2%to 10% by weight, the maltodextrin may be contained in an amount of 2%to 10% by weight, the skim milk powder may be contained in an amount of0.5% to 2% by weight, and the starch may be contained in an amount of0.1% to 1% by weight in the composition. In addition, the excipient maybe contained in an amount of 75% to 95% by weight or 85% to 95% byweight based on the total weight of the composition.

Further, the method for preparing the composition may include mixing thestrains of Lactobacillus plantarum CJLP475 and the Lactobacillusplantarum CJLP17 themselves, a lysate thereof, a culture thereof, aconcentrate thereof, or a dried product thereof with an additive. Theadditive may be the above-mentioned cryoprotectant or excipient.

The composition may be used for a food, a functional food, a feed, afeed additive, a cosmetic composition, or a pharmaceutical composition.

The composition may be used for enhancing immunity.

The composition may be used for providing an antiviral activity.

In another aspect, there is provided a probiotic composition includingthe composition of the present disclosure as an active ingredient.

Probiotics are fixed on the walls of the digestive tract in theintestines to prevent the establishment of harmful bacteria and inhibitthe proliferation of viruses. In addition, the beneficial digestiveenzymes produced by probiotics promote growth by facilitating theabsorption and utilization of nutrients.

A method for preparing the probiotic composition may include preparingeach of a composition including a Lactobacillus plantarum CJLP475 straindeposited under Accession No. KCCM12287P and a composition including aLactobacillus plantarum CJLP17 strain deposited under Accession No.KCCM12249P; and mixing the composition including the CJLP475 strain andthe composition including the CJLP17 strain.

In one embodiment, the preparation method may include adding an additiveto at least one of the compositions above, such as the compositionincluding the CJLP475 strain, the composition including the CJLP17strain, or the probiotic composition

In another embodiment, the additive may be a cryoprotectant, and themethod may further include a freeze-drying step after adding theadditive.

Herein, the bacteria in the freeze-dried composition may be in a livestate.

In still another embodiment, the preparation method may includepreparing a predetermined amount of the composition into a package afterthe mixing step.

Herein, in the packaging step, the composition may be prepared into apackage such that the total amount of strains including the CJLP475strain and the CJLP17 strain is 10⁶ CFU/g or more.

In still another aspect of the present disclosure, there is provided afeed or a feed additive including the composition.

The composition above is as described above. Specifically, thecomposition of the present disclosure may be added to a feed additive ora feed composition including the feed additive for the purpose ofpromoting growth, reducing diarrhea incidence, and inhibiting viralactivity.

As used herein, the term “feed additive” refers to substances added to afeed for the purpose of providing various effects, such as supplementingnutrients and preventing weight loss, promoting digestibility ofcellulose in the feed, improving milk quality, preventing reproductivedisorders and improving pregnancy rate, and preventing ahigh-temperature stress during the summer season. The feed additive ofthe present disclosure belongs to a supplementary feed according to theControl of Livestock and Fish Feed Act and may further include mineralpreparations such as sodium hydrogen carbonate, bentonite, magnesiumoxide, complex minerals, and trace minerals including zinc, copper,cobalt, and selenium; vitamins such as carotene, vitamin E, vitamins A,D, E, nicotinic acid, and vitamin B complex; amino acid protectiveagents such as methionine and lysine; fatty acid protective agents suchas fatty acid calcium; and live bacteria and yeast preparations such asprobiotics (lactic acid bacteria), yeast culture, and fungus fermentedproduct.

As used herein, the term “feed” refers to any natural or artificialdiet, a single meal, or the like, or a component of the single meal,which an animal eats, ingests and digests or which is suitable foreating, ingestion, and digestion. The feed including the composition forpreventing or treating a metabolic disease according to the presentdisclosure as an active ingredient may be prepared into various forms offeeds known in the art, and may specifically include a concentratedfeed, a crude feed, and/or a specialty feed.

The subjects to be raised may include any organism that can ingest thefeed of the present disclosure, and may include pigs for the purpose ofthe present disclosure.

The content of the composition in the feed composition according to thepresent disclosure may be properly controlled depending on the kind andage of a subject to be applied, application forms, desired effects, andthe like. For example, the composition may be contained in an amount of0.01% to 20% by weight, 0.01% to 15% by weight, 0.01% to 10% by weight,0.01% to 5% by weight, 0.01% to 1% by weight, 1% to 20% by weight, 1% to15% by weight, 1% to 10% by weight, 1% to 5% by weight, 5% to 20% byweight, 5% to 15% by weight, 5% to 10% by weight, 10% to 20% by weight,10% to 15% by weight, or 15% to 20% by weight, but is not limitedthereto.

For administration, the feed composition of the present disclosure mayfurther include a mixture of one or more of an organic acid such ascitric acid, fumaric acid, adipic acid, lactic acid, and the like;phosphate such as potassium phosphate, sodium phosphate, polyphosphate,and the like; and a natural antioxidant such as polyphenol, catechin,tocopherol, vitamin C, green tea extract, chitosan, tannic acid, and thelike. If necessary, other typical additives such as an anti-influenzaagent, a buffer, a bacteriostatic agent, and the like may be added.Further, a diluent, a dispersing agent, a surfactant, a binder or alubricant may be additionally added to formulate the composition into aninjectable preparation such as an aqueous solution, a suspension, anemulsion, and the like, a capsule, a granule, or a tablet.

Further, the feed composition of the present disclosure may be usedtogether with a nutrient supplement, a growth accelerator, adigestion-absorption accelerator, and a prophylactic agent, in additionvarious auxiliaries such as amino acids, inorganic salts, vitamins,antioxidants, antifungal agents, antimicrobial agents, and the like, asauxiliary components, and the main ingredients including vegetableprotein feeds such as pulverized or fragmented wheat, barley, corn, andthe like, animal protein feeds such as blood meal, meat meal, fish meal,and the like, animal fat and vegetable fat.

When the feed composition of the present disclosure is used as a feedadditive, the feed composition may be added either alone or incombination with other components, and may be appropriately usedaccording to a conventional method. The feed composition may be preparedin the administration form of an immediate release or a sustainedrelease formulation, in combination with a non-toxic, pharmaceuticallyacceptable carrier. The carrier may be a non-naturally occurringsubstance or a naturally occurring substance, but is not limitedthereto. In another embodiment, the carrier may be a substance that doesnot naturally contact with the Lactobacillus plantarum CJLP475 strainand the Lactobacillus plantarum CJLP17 strain, or a substance that isnot naturally contained simultaneously with the two strains, but is notlimited thereto. The edible carrier may be corn starch, lactose,sucrose, or propylene glycol. A solid carrier may be in theadministration form of tablets, powders, troches, and the like, and aliquid carrier may be in the administration form of syrups, liquidsuspensions, emulsions, solutions, and the like. Further, theadministration agent may include a preservative, a lubricant, a solutionaccelerator, or a stabilizer, and may also include other agents forimproving inflammatory diseases and substances useful for the preventionof virus.

The feed composition according to the present disclosure may be mixedwith a feed in an amount of about 10 g to 500 g, specifically 10 g to100 g per 1 kg, based on the dry weight of the feed. After beingcompletely mixed, the feed composition may be provided as mash, or maybe further subjected to a pelletizing, extensification, or extrusionprocess, but is not limited thereto.

In still further another aspect of the present disclosure, there isprovided a food or a functional food including the composition.

Specifically, the composition of the present disclosure may be added tofood for the purpose of promoting growth, promoting immunity, reducingdiarrhea incidence, and inhibiting viral activity. The composition is asdescribed above. The food may include a sitologically acceptablecarrier. The carrier may be a non-naturally occurring substance or anaturally occurring substance, but is not limited thereto. In anotherembodiment, the carrier may be a substance that does not naturallycontact with the Lactobacillus plantarum CJLP475 strain and theLactobacillus plantarum CJLP17 strain, or a substance that is notnaturally contained simultaneously with the two strains, but is notlimited thereto.

The food of the present disclosure includes all forms of functionalfoods, nutritional supplements, health foods and food additives, andthese types of food may be prepared into various forms according toconventional methods.

When the composition is used as a food additive, the composition may beadded either alone or used in combination with other foods or foodingredients, and may be appropriately used according to a conventionalmethod. The amount of mixed active ingredients may appropriately bedetermined depending on the purpose of use (prevention, health, ortherapeutic treatment). In general, at the time of preparing a food ordrink, the composition is added in an amount of 0.0001% to 1% by weight,specifically 0.0001% to 0.1% by weight based on a raw materialcomposition including the composition. However, in the case of long-termadministration for health and hygiene purposes or for the purpose ofcontrolling health, the amount may be less than the above-describedrange

There is no particular limitation on the type of the food. Examples offoods to which the composition can be added include meats, sausages,bread, chocolates, candies, snacks, confectionaries, pizzas, instantnoodles, other noodles, gums, dairy products including ice creams,various kinds of soup, beverages, teas, drinks, alcoholic drinks,vitamin complexes, and the like, and all health functional foods in theordinary sense are included.

The health drink composition of the present disclosure may furthercontain, as additional components, various flavoring agents or naturalcarbohydrates, as in conventional drinks. The aforementioned naturalcarbohydrates may include monosaccharides such as glucose, fructose, andthe like; disaccharides such as maltose, sucrose, and the like;polysaccharides such as dextrin, cyclodextrin, and the like; and sugaralcohols such as xylitol, sorbitol, erythritol, and the like. Naturalsweetening agents such as thaumatin, a stevia extract, and the like; andsynthetic sweetening agents such as saccharin, aspartame, and the likemay be used as the sweetening agent. A ratio of the additionalcomponents may be in a range of 0.01 to 0.04 parts by weight,specifically 0.02 to 0.03 parts by weight based on 100 parts by weightof the composition of the present disclosure.

In addition to the aforementioned components, the composition of thepresent disclosure may contain various nutritional supplements,vitamins, electrolytes, flavoring agents, coloring agents, pectic acidand salts thereof, alginic acid and salts thereof, organic acids,protective colloidal thickening agents, pH control agents, stabilizingagents, preservatives, glycerin, alcohols, carbonating agents used incarbonated drinks, and the like. The ratio of such additives is notimportant, but is generally chosen in a range of 0.01 to 0.1 parts byweight, based on 100 parts by weight of the composition of the presentdisclosure. Moreover, the composition of the present disclosure mayinclude pulp for preparing a natural fruit juice, a fruit juice drink ora vegetable drink. The ratio of such pulp is not important, but isgenerally chosen in a range of 0.01 to 10 parts by weight, based on 100parts by weight of the composition of the present disclosure. Suchcomponents may be used alone or in combination.

In still further another aspect of the present disclosure, there isprovided a cosmetic including the composition.

Specifically, the composition of the present disclosure has ananti-inflammatory effect and an antiviral activity-inhibiting effectthrough immunity stimulation, and thus can be used as a cosmetic. Thecomposition is as described above.

When the composition according to the present disclosure is used as acosmetic, the composition may be prepared into various cosmetics in theconventional formulations known in the field of cosmetics. Uponpreparation into each formulation, it may be prepared by adding acarrier or an excipient which is acceptable and necessary in themanufacture of the cosmetics for each formulation. The carrier may be anon-naturally occurring substance or a naturally occurring substance,but is not limited thereto. In another embodiment, the carrier may be asubstance that does not naturally contact with the Lactobacillusplantarum CJLP475 strain and the Lactobacillus plantarum CJLP17 strain,or a substance that is not naturally contained simultaneously with thetwo strains, but is not limited thereto.

In still further another aspect of the present disclosure, there isprovided a pharmaceutical including the composition. Specifically, thecomposition of the present disclosure has an anti-inflammatory effectand an antiviral activity-inhibiting effect by promoting growth,reducing diarrhea incidence and stimulating immunity, and thus can beused as a pharmaceutical. The composition is as described above.

When the composition according to the present disclosure is used as apharmaceutical, the composition may be prepared into a conventionalpharmaceutical formulation known in the art. The pharmaceutical mayspecifically be prepared into formulations for oral administration suchas liquids, suspensions, powder, granules, tablets, capsules, pills, orextracts. Upon preparation into each formulation, it may be prepared byadding a carrier or an excipient which is acceptable and necessary inthe manufacture of the cosmetics for each formulation. The carrier maybe a non-naturally occurring substance or a naturally occurringsubstance, but is not limited thereto. In another embodiment, thecarrier may be a substance that does not naturally contact with theLactobacillus plantarum CJLP475 strain and the Lactobacillus plantarumCJLP17 strain, or a substance that is not naturally containedsimultaneously with the two strains, but is not limited thereto.Typically, when the composition is prepared into a formulation for oraladministration, at least one carrier selected from a diluent, alubricant, a binder, a disintegrating agent, a sweetener, a stabilizerand a preservative may be used, and at least one additive selected froma flavoring agent, a vitamin and an antioxidant may be used. Anypharmaceutically acceptable excipient or additive may be used.Specifically, it is possible to use lactose, corn starch, soybean oil,microcrystalline cellulose or mannitol as the diluent; magnesiumstearate or talc as the lubricant; and polyvinyl pyrrolidone orhydroxypropyl cellulose as the binder. In addition, it is possible touse calcium carboxymethylcellulose, sodium starch glycolate, polacrilinpotassium or crospovidone as the disintegrating agent; white sugar,fructose, sorbitol or aspartame as the sweetener; sodiumcarboxymethylcellulose, β-cyclodextrin, white wax or xanthan gum as thestabilizer; and methyl paraoxybenzoate, propyl paraoxybenzoate orpotassium sorbate as the preservative.

In still further another aspect of the present disclosure, there isprovided a step of administering the composition of the presentdisclosure to a subject. The composition and the subject are asdescribed above.

The subject may exhibit the efficacy of the composition, i.e.,immune-enhancing activity, antiviral activity, and the like, by theadministration.

The dosage for the administration may be, but is not limited to, 10⁶CFU/day or more, 10⁷ CFU/day or more, 10⁸ CFU/day or more, 10⁹ CFU/dayor more, 10¹⁰ CFU/day or more or 10¹¹ CFU/day or more based on themixture of the Lactobacillus plantarum CJLP475 strain and theLactobacillus plantarum CJLP17 strain.

In still further another aspect of the present disclosure, there isprovided a method for enhancing immunity of a subject, includingadministering the composition to a subject in need. The composition andthe subject are as described above.

As used therein, the term “administration” means introducing thecomposition of the present disclosure to a subject by any appropriatemethod, and the administration route of the composition may includevarious routes such as oral or parenteral administration as long as itcan reach the target tissue.

In still further another aspect of the present disclosure, there isprovided a method for preventing or treating virus-infected disease of asubject, including administering the composition to a subject in need.The composition and the subject are as described above.

As used herein, the term “prevention” or “preventing” means all actionsthat are intended to inhibit, suppress, or delay a virus-infecteddisease by administration of the composition of the present disclosure.Further, as used herein, the term “treatment” or “treating” means allactions that are intended to ameliorate or beneficially change a symptomof a virus-infected disease by administration of the composition of thepresent disclosure.

DETAILED DESCRIPTION OF EMBODIMENTS

Hereinafter, the present disclosure will be described in more detail byway of Examples. However, these Examples are given for illustrativepurposes only, and the scope of the invention is not intended to belimited by these Examples.

Example 1: Separation and Selection of Strains

1-1. Sample Collection and Separation of Lactobacillus Plantarum CJLP475Strain

Lactobacillus plantarum CJLP475 strains isolated from soy sauce weresmeared on a solid MRS medium (Difco, USA) containing 1.5% agar andincubated at 37° C. for 24 hours. The strains isolated from each samplewere purely isolated by transferring them to a fresh medium, and thethus-isolated strains were stored in a nutrient medium supplemented with20% glycerol at −70° C. or below. As a result, a total of 1,552 strainswere collected, and strains having excellent antiviral activity wereselected through the following examples.

As a result of the 16S rDNA sequencing analysis, the Lactobacillusplantarum CJLP475 strain showed the highest homology (99.9%) with theLactobacillus plantarum standard strain (NBRC1589, GenBank AccessionNumber AB326351) and thus was identified as Lactobacillus plantarum andnamed “Lactobacillus plantarum CJLP475”, and deposited at the KoreanCulture Center of Microorganisms, an International Depositary Authority,under Budapest Treaty on Jul. 11, 2018, with Accession No. KCCM12287P.The analyzed 16S rDNA nucleotide sequence of the Lactobacillus plantarumCJLP475 is represented by SEQ ID NO: 1.

Meanwhile, the Lactobacillus plantarum CJLP475 strain was found to begram-positive as a result of the Gram staining. Further, in order toanalyze the biochemical characteristics, sugar fermentation patterns ofthe strain were analyzed by the API 50 CHL system (biomerieux Vitek,Inc., France) (Table 1).

TABLE 1 Analysis of Sugar Fermentation Patterns of Lactobacillusplantarum CJLP475 Name of strain CJLP475 Name of strain CJLP475 Control− Esculin + Glycerol − Salicin + Erythritol − Cellobiose + D-Arabinose −Maltose + L-Arabinose + Lactose + Ribose + Melibiose + D-Xylose −Saccharose + L-Xylose − Trehalose + Adonitol − Inulin − βMethyl-xyloside − Melezitose + Galactose + D-Raffinose + D-Glucose +Amidon − D-Fructose + Glycogen − D-Mannose + Xylitol − L-sorbose −β-Gentiobiose + Rhamnose + D-Turanose + Dulcitol − D-Lyxose − Inositol −D-Tagatose − Mannitol + D-Fucose − Sorbitol + L-Fucose − αMethyl-D-mannoside + D-Arabitol − α Methyl-D-glucoside − L-Arabitol − NAcetyl glucosamine + Gluconate + Amygdaline + 2-ceto-gluconate −Arbutin + 5-ceto-gluconate − +: Positive, −: Negative

1-2. Sample Collection and Separation of Lactobacillus Plantarum CJLP17Strain

Samples of feces and colostrum of sows and piglets were collected fromthe domestic farms where chronic outbreaks of PED virus had occurred.The thus-collected samples were subjected to a serial dilution, smearedon a solid MRS and BHI medium, and incubated at 37° C. for 48 hours. Thestrains isolated from each sample were purely isolated by transferringthem to a fresh medium, and the thus-isolated strains were stored in anutrient medium supplemented with 20% glycerol at −70° C. or below. As aresult, a total of 1,552 strains were collected, and strains havingexcellent antiviral activity were selected through the followingexamples. As a result of 16S rDNA sequencing analysis, the Lactobacillusplantarum CJLP17 strain showed the highest homology (99.9%) with theLactobacillus plantarum standard strain (NBRC15891, GenBank AccessionNumber AB326351) and was thus identified as Lactobacillus plantarum andnamed “Lactobacillus plantarum CJLP17”, and deposited at the KoreanCulture Center of Microorganisms, an International Depositary Authority,under Budapest Treaty on Apr. 13, 2018, with Accession No. KCCM12249P.The analyzed 16S rDNA nucleotide sequence of Lactobacillus plantarumKCCM12249P is represented by SEQ ID NO: 2.

Meanwhile, the Lactobacillus plantarum CJLP17 strain was found to begram-positive as a result of the Gram staining. Further, in order toanalyze the biochemical characteristics, sugar fermentation patterns ofthe strain were analyzed by the API 50 CHL system (biomerieux Vitek,Inc., France) (Table 2).

TABLE 2 Analysis of Sugar Fermentation Patterns of Lactobacillusplantarum CJLP17 Name of strain CJLP17 Name of strain CJLP17 Control −Esculin − Glycerol − Salicin + Erythritol − Cellobiose + D-Arabinose −Maltose + L-Arabinose + Lactose + Ribose + Melibiose + D-Xylose −Saccharose + L-Xylose − Trehalose + Adonitol − Inulin − βMethyl-xyloside − Melezitose + Galactose + D-Raffinose − D-Glucose +Amidon − D-Fructose + Glycogen − D-Mannose + Xylitol − L-sorbose −B-Gentiobiose + Rhamnose − D-Turanose + Dulcitol − D-Lyxose − Inositol −D-Tagatose − Mannitol + D-Fucose − Sorbitol + L-Fucose − αMethyl-D-mannoside + D-Arabitol − α Methyl-D-glucoside − L-Arabitol − NAcetyl glucosamine + Gluconate + Amygdaline + 2-ceto-gluconate −Arbutin + 5-ceto-gluconate − +: Positive, −: Negative

Example 2: Assessment of Acid-Resistance and Bile-Resistance of Strains

In order to select the strains that can be used as probiotics,acid-resistance and bile-resistance of the obtained strains wereassessed.

An artificial gastric juice medium was prepared for the assessment ofacid-resistance. More specifically, the artificial gastric juice mediumwas prepared by adding pepsin to a liquid MRS medium so as to adjust thepH to 2.5, followed by sterilization.

The strains of Example 1 were subjected to a static culture in a liquidMRS medium at 37° C. for 18 hours after the second subculture. 1% of thepre-incubated strains were inoculated to the artificial gastric juicemedium and subjected to a static culture at 37° C., and the culturebroth was sampled at 0 hour and 3 hours. The sampled culture broth wasserial diluted and smeared on the solid MRS medium, and incubated at 37°C. for 48 hours to measure viable cell count.

An artificial bile medium was prepared for the assessment ofbile-resistance. More specifically, the artificial bile medium wasprepared by adding 0.5% oxgall((bull bile) to a liquid MRS medium,followed by sterilization.

The strains of Example 1 were subjected to a static culture in a liquidMRS medium at 37° C. for 18 hours after the second subculture. 1% of thepre-incubated strains were inoculated to the artificial bile medium andsubjected to a static culture at 37° C., and the culture broth wassampled at 0 hour and 24 hours. The sampled culture broth was serialdiluted and smeared on the solid MRS medium, and incubated at 37° C. for48 hours to measure viable cell count.

Through the above assessments, the strains having the highestacid-resistance and bile-resistance were selected and designated asLactobacillus plantarum CJLP475 and Lactobacillus plantarum CJLP17. Inorder to compare the acid-resistance and bile-resistance of theLactobacillus plantarum CJLP475 strain and the Lactobacillus plantarumCJLP17 strain with those of the conventionally known strains, theacid-resistance and the bile-resistance of the Lactobacillus plantarumstandard strain (KCCM12116) obtained from the Korean Culture Center ofMicroorganisms were assessed in the same manner as the above method.

TABLE 3 Acid-Resistance Assessment (Unit: CFU/mL) 0 hr 3 hrsLactobacillus plantarum CJLP17 1.1 × 10⁶ 4.0 × 10⁶ (KCCM12249P)Lactobacillus plantarum CJLP475 1.1 × 10⁶ 1.1 × 10⁷ (KCCM12287P)Lactobacillus plantarum 2.3 × 10⁷ 1.3 × 10⁷ (KCCM12116)

TABLE 4 Bile-Resistance Assessment (Unit: CFU/mL) 0 hr 24 hrsLactobacillus plantarum CJLP17 1.3 × 10⁶ 1.1 × 10⁷ (KCCM12249P)Lactobacillus plantarum CJLP475 1.3 × 10⁸ 1.1 × 10⁷ (KCCM12287P)Lactobacillus plantarum 2.1 × 10⁷ 1.6 × 10⁶ (KCCM12116)

According to Tables 3 and 4 above, the number of cells of theLactobacillus plantarum standard strain (KCCM12116) in the artificialgastric juice medium and the artificial bile medium was decreased. As aresult, it can be seen that not all commonly known Lactobacillusplantarum have acid-resistance and bile-resistance.

Meanwhile, in the acid-resistance assessment, the number of cells in theLactobacillus plantarum CJLP475 strain and the Lactobacillus plantarumCJLP17 strain was rather increased, indicating that the strains haveexcellent acid-resistance.

In the bile-resistance assessment, the number of cells in theLactobacillus plantarum CJLP17 strain was increased, and theLactobacillus plantarum CJLP475 strain had a smaller decrease in thenumber of cells as compared with the Lactobacillus plantarum standardstrain (KCCM12116), indicating that both strains have excellentbile-resistance.

In addition, since each strain has excellent acid-resistance andbile-resistance, it can be implied that the composition including bothstrains has excellent acid-resistance and bile-resistance.

Example 3: Assessment of Safety of Strains

3-1. Confirmation of Hemolytic Activity of Strains

β-Hemolysis is a phenomenon in which phospholipids supplied by red bloodcells are hydrolyzed by phospholipid enzymes produced from harmfulbacteria, resulting in hemolysis of red blood cells. In order todetermine the hemolytic activity of the Lactobacillus plantarum CJLP475strain and the Lactobacillus plantarum CJLP17 strain, blood agar plates(sheep blood 5% agar, Hanilkomed, Korea) were used. Each strain wasstreaked into the prepared blood agar plates and incubated at 37° C. for24 hours to confirm the hemolysis.

As a result, as shown in FIG. 1, it was confirmed that the Lactobacillusplantarum CJLP475 strain and the Lactobacillus plantarum CJLP17 straindid not show hemolysis. These results suggest that they do not act asharmful bacteria in vivo.

3-2. Assessment of Antibiotic Susceptibility

The Lactobacillus plantarum CJLP475 strain and the Lactobacillusplantarum CJLP17 strain were independently inoculated into a liquid MRSmedium and subjected to a static culture at 37° C. for 24 hours. Thethus-cultured bacteria were soaked in sterilized cotton swabs andsmeared on a solid Mueller Hinton II medium (Difco), and then antibioticdiscs were placed on the medium and incubated at 37° C. for 24 hours.Ampicillin, clindamycin, gentamicin, kanamycin, erythromycin,ampicillin/sulbactam, chloramphenicol, and streptomycin discs (Oxoid,UK) were used as antibiotic discs for the antibiotic test.

As a result of the antibiotic susceptibility test of the Lactobacillusplantarum CJLP475 strain and Lactobacillus plantarum CJLP17 strain, eachstrain was not resistant to the above antibiotics (Table 5). Therefore,it can be found that even if the Lactobacillus plantarum CJLP475 strainand the Lactobacillus plantarum CJLP17 strain are used inpharmaceuticals, health functional foods, feed additives, etc., problemsthat may arise with respect to the resistance and environmental problemsare less likely to occur, considering that they have no resistance toantibiotics.

TABLE 5 Inhibition of Bacterial Growth According to Antibiotics Radiusof Growth Inhibition Area Centered Around Antibiotics (mm) AntibioticsCJLP475 CJLP17 Amp10 (Ampicillin) 7.5 7.5 C30 (Clindamycin) 7 7 CN120(Gentamicin) 5 5 K30 (Kanamycin) 1.5 1.5 E15 (Erythromycin) 12 12 SAM20(Ampicillin/Sulbactam) 7 7 S10 (Chloramphenicol) 3.5 3.5 DA2(Streptomycin) 4.5 4.5

Example 4: Assessment of Cytotoxicity

In order to investigate the effect of the strains on the survival ofcells, the MTS assay was carried out using(3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,(promega, USA) to assess the level of cytotoxicity on IPEC-J2 cells(intestinal pig epithelium cells). Each cell was incubated on a 96-wellcell culture plate and treated with a strain mixture, in which theLactobacillus plantarum CJLP475 and Lactobacillus plantarum CJLP17 weremixed in a ratio of 1:1, at different concentrations from 10⁵ CFU/mL to10⁷ CFU/mL. After 24 hours, the MTS solution was added to the cellculture broth, and the cells were incubated for 2 hours, and the cellsurvival rate (%) was calculated by measuring the absorbance at 490 nmwith a microplate reader.

As a result, as shown in FIG. 2, when the cells were treated at threedifferent concentrations, it was confirmed that cell death was hardlyobserved at concentrations of 10⁷ CFU/mL or below. Accordingly, it canbe seen that the composition substantially shows no cytotoxicity atconcentrations of 10⁷ CFU/mL or below.

Example 5: Inhibitory Effect Against Viral Infection

In order to measure the inhibitory effect of the composition includingthe Lactobacillus plantarum CJLP475 strain and the Lactobacillusplantarum CJLP17 strain against viral infection, porcine epidemicdiarrhea virus (PEDV) was prepared. Specifically, the virus wasproliferated in Vero cells (CCL-81, kidney epithelial cells extractedfrom Chlorocebus), and MEM (Eagle's Minimum Essential Medium, Gibco BRL,USA), heat-inactivated 10% FBS (fetal bovine serum, v/v) and 1% (v/v)penicillin/streptomycin were as the media for culturing Vero cells. TheVero cells were incubated as a monolayer, washed twice with the media,and then all the solutions were removed. The virus was mixed at 0.1 MOI(multiplicity of infection) level in FBS-free MEM containing trypsintreated with 5 μg/mL TPCK (N-tosyl-L-phenylalanine chloromethyl ketone),treated with a minimal volume of the prepared culture cells, and thenincubated in a 37° C. cell incubator containing 5% CO₂ for 2 to 3 days.

The viral infection was determined by the formation of virus syncytia.When a virus syncytium was formed, the virus culture broth was collectedwithin 3 to 6 hours, and the cells were removed using a centrifuge andstored at −80° C. For the calculation of the infection titer of thevirus, the Vero cells were incubated in a 96-well plate at a density of2×10⁴ cell/0.1 mL, and the cells were washed with PBS. Subsequently, thecells were added with a culture broth, in which the virus was subjectedto a 2-fold serial dilution, and incubated for 24 to 48 hours to confirmviral infection, and the virus titer was calculated by the Reed & Muenchmethod.

In order to measure the inhibitory effect of the composition includingthe Lactobacillus plantarum CJLP475 strain and the Lactobacillusplantarum CJLP17 strain against viral infection, the composition, andperipheral blood mononuclear cells (PBMC) and lymphocytes in mesentericlymph node (mLN) extracted from 21-day-old weaned piglets were reactedfor 20 to 24 hours, and then independently treated on the 96-well cellculture plate, in which IPEC-J2 was incubated, and incubated in a 37° C.cell incubator containing 5% CO₂ for 2 to 4 hours. The PED virus (SM98or KPEDV9) at a dose of 100 TCID₅₀/mL (50% of tissue cell infectiousdose) was aliquoted to each plate and incubated for 48 hours. In orderto confirm the viral infection, the cell culture plate was fixed withmethanol after completion of the culture, stained with crystal violet,and then the wells in which the cells were denatured, were examined witha microscope, thereby confirming the viral infection.

TABLE 6 Inhibitory Effect against PED virus infection of Lactobacillusplantarum CJLP475, Lactobacillus plantarum CJLP17, and Mixture thereofby Activated Immune Cells mLN PBMC PEDV PEDV Negative Control − −CJLP475 + ++ CJLP17 + ++ CJLP17 and CJLP475 ++ ++ ++: CompleteInhibition, +: Partial Inhibition, −: Infected

As a result, as shown in Table 6 and FIG. 3, when the compositionincluding both the Lactobacillus plantarum CJLP475 strain and theLactobacillus plantarum CJLP17 strain was treated together with porcineimmune cells, it can be judged that the immune cells activated by thecomposition can inhibit PED virus infection in the intestinal pigepithelial cells (IPEC-J2).

Considering that the total number of strains for each of the threeexperimental groups (CJLP475, CJLP17, CJLP17 and CJLP475) in Table 6were the same, the inhibitory effect of the composition including boththe Lactobacillus plantarum CJLP475 strain and the Lactobacillusplantarum CJLP17 strain against PED virus infection was significantlysuperior compared to the inhibitory effect of the compositions includingeach of the CJLP475 strain and the CJLP17 strain against the PED virusinfection.

Example 6: Confirmation of Effect on Immunity and Colostrum of Sows

Since the antiviral effect of the composition including theLactobacillus plantarum CJLP475 strain and the Lactobacillus plantarumCJLP17 strain was confirmed in Example 5, an experiment was carried outin order to confirm the effect thereof on the immunity and colostrum ofsows when the composition was fed to the sows, as follows: fifteen sowsat 6 weeks before delivery in the same delivery period were selectedrandomly for each experimental group from the farms in which no PEDvirus had occurred. All of the sows used in the experiment wereinoculated with the PED virus vaccine at 8 weeks and 3 weeks beforedelivery. The experiment was carried out in a total of two experimentalgroups, i.e., a control group in which no strain was fed and a group fedwith the composition including the Lactobacillus plantarum CJLP475strain and the Lactobacillus plantarum CJLP17 strain. The feed was givenin the form of common crumble feeds without antibiotics, and water wasgiven to be consumed freely. The Lactobacillus plantarum CJLP475 strainand Lactobacillus plantarum CJLP17 strain were produced in the form offreeze-dried powder and stored in a refrigerator, and then added on topof the feed when given to the sows so that each sow was fed with thecomposition in an amount of 10¹⁰ CFU or more per day. The sows were fedfor a total of 6 weeks, and in order to confirm the effect of thestrains on the immunity and colostrum of the sows, the increase rate ofvaccine-specific IgG antibody and the increase rate of vaccine-specificIgA antibody in sow serum, and the neutralizing antibody titer incolostrum were measured, and the results are shown in FIGS. 4, 5, and 6,respectively.

As a result of the experiment, the compositions including theLactobacillus plantarum CJLP475 strain and the Lactobacillus plantarumCJLP17 strain increased the vaccine-specific IgG and IgA in sow serumcompared to the control group. Further, when the change in theneutralizing antibody titer in the colostrum during delivery accordingto the feeding of the composition was examined, it was confirmed thatthe composition including the Lactobacillus plantarum CJLP475 strain andthe Lactobacillus plantarum CJLP17 strain showed a higher neutralizingantibody titer than the control group.

Example 7: Effect of Improving Immunity of Piglets According to Feedingof the Composition in Sows

In order to confirm the effect of the composition including theLactobacillus plantarum CJLP475 strain and the Lactobacillus plantarumCJLP17 strain on the immunity of piglets born from the sows fed with thecomposition, an experiment was carried out using sows as in Example 6,and further, blood was collected from the piglets born from the sows 4days after given colostrum to carry out an immunological analysis usingan IgG ELISA kit. FIG. 7 shows the effect of the composition feeding onthe immunity of piglets born from the sows for a total of 6 weeks.

As a result, the content of vaccine-specific IgG in piglet serum wasfound to be higher in the group fed with the composition including theLactobacillus plantarum CJLP475 strain and the Lactobacillus plantarumCJLP17 strain than in the control group (FIG. 7).

Such results indicate that the piglets received high vaccine-specificIgG from the colostrum of the sows and thus, it can be found that theimmunity of the piglets can be improved by feeding the sows with thecomposition including the Lactobacillus plantarum CJLP475 strain and theLactobacillus plantarum CJLP17 strain.

Example 8: Effect of Challenge Test on Piglets Born from Sows Fed withthe Composition

In order to confirm the effect on the mortality of piglets when thepiglets born from the sows fed with composition including theLactobacillus plantarum CJLP475 strain and the Lactobacillus plantarumCJLP17 strain were infected with PED virus, an experiment was carriedout using sows as in Example 6. Since this experiment required achallenge test on piglets with PED virus, the experimental groups wereminimized according to the advice of the institutional animal care anduse committee, in such a manner that the experimental group containingthe single strain was not tested and only the group containing thecomposition including the Lactobacillus plantarum CJLP475 strain and theLactobacillus plantarum CJLP17 strain was tested.

Twenty 4-day-old piglets after delivery from the sows fed with thecomposition including the Lactobacillus plantarum CJLP475 strain and theLactobacillus plantarum CJLP17 strain were selected for each treatmentgroup and challenged with PED virus (PEDv QIAP 1401, 100 LD₅₀%/ml). Thebody temperature and diarrhea score were measured daily for a total of 7days after the challenge (0-3, 0: normal stool, 1: pale stool, 2: normaldiarrhea, 3: severe diarrhea). The piglets were given milk substituteand water to be consumed freely, and each piglet was fed with thecomposition including the Lactobacillus plantarum CJLP475 strain and theLactobacillus plantarum CJLP17 strain in an amount of 1×10¹⁰ CFU per dayby mixing with the milk substitute. When the piglets died, an autopsywas performed, and the jejunum and colon were sampled, fixed withneutralized formalin, embedded by treating with paraffin, andmicro-sectioned, and then the tissue sections were de-paraffinized andhydrated. After H & E staining, the ratio of the height of the villi andthe depth of the crypts was measured to examine the V/C ratio. Thenumber of goblet cells per mm² was confirmed by sampling the mucosa ofthe colon. In addition, blood was collected from each subject and theserum was separated therefrom to compare the degree of inflammationinhibition for each treatment group. In this regard, the level ofTGF-beta, which is a typical inflammation-inhibiting factor, wasmeasured using ELISA (R&D Systems, USA), and the results of thechallenge test are shown in FIGS. 8 to 13.

TGF-beta in piglet serum was found to be higher in the composition-fedgroup than in the control group. Therefore, it was confirmed that theanti-inflammatory reaction was further activated in the group fed withthe composition including the Lactobacillus plantarum CJLP475 strain andthe Lactobacillus plantarum CJLP17 strain (FIG. 8).

The V/C ratio of the piglet jejunum was higher in the composition-fedgroup than in the control group, and thus it was confirmed that theremoval of the villi by the virus was alleviated as compared with thecontrol group (FIG. 9).

The goblet cells in the piglet colon were present in a higher content inthe composition-fed group than the control group, and thus it wasconfirmed that the intestinal mucosa-forming inhibition caused by thePED virus infection was significantly reduced (FIG. 10).

As a result of examining the change in the body temperature of thepiglets after PED virus challenge, it was confirmed that the bodytemperature was maintained within the normal range in thecomposition-fed group as compared to the control group. In particular,at 5, 6 and 7 dpi, the change in the body temperature of the pigletsborn from the sows fed with the composition significantly maintained themean body temperature as compared to the control (FIG. 11).

As a result of confirming the diarrhea score (clinical significancescore) of the piglet caused by the PED virus, the diarrhea score in theexperimental group fed with the composition was significantly lower thanthat of the control group at 6 and 7 dpi (FIG. 12).

The survival rate of the piglets was observed until 7 days after the PEDvirus challenge. As a result, the survival rate of the control group was55%, and the survival rate of the piglets born from the sows fed withPED virus was 65% when infected with PED virus, thereby showing moresuperior antiviral effect compared to the control group (FIG. 13).

Based on the above results, it was confirmed that the mortality rate ofthe piglets was reduced upon infection with the virus, because theimmunity of the sows and the antibodies in the colostrum were improvedby feeding the sows with the composition, and thus had an effect on theimmunity of the piglets.

Example 9: Preparation of Probiotics Including Lactobacillus plantarumCJLP475 Strain and the Lactobacillus plantarum CJLP17 Strain

The probiotic Lactobacillus plantarum CJLP475 strain and the probioticLactobacillus plantarum CJLP17 strain identified in Example 1 wereproduced on a mass-scale and freeze-dried to make probiotics suitablefor use as a raw material of pharmaceuticals, foods, feeds, feedadditives or cosmetics.

For production of each strain, each strain was incubated in a liquid MRSmedium (Difco) at 37° C. for 18 hours while adjusting the pH to 6.0 witha 25% NaOH solution, followed by harvesting the strains bycentrifugation. Each of the harvested strains was frozen at −40° C. with5% dextrin and 10% skim milk serving as cryoprotectants, dried at 37° C.and powdered using a mixer. Each of the powdered live strains was mixedeach other to adjust the number of strains to a desired level. Themixture was mixed with a suitable amount of an excipient such asglucose, lactose and skim milk for storage, and packaged in a sealablealuminum pouch.

The probiotics can be applied to various fields in accordance with aconventional method in the art, such as pharmaceuticals, foods, feeds,cosmetics and the like. For example, the prepared probiotics may bemixed with grain powder used as a raw material for feeds to be used asprobiotics for feeds, may be mixed with an excipient or additive to beused as probiotics for pharmaceuticals in the form of tablets, capsulesand for foods, or may be mixed in a predetermined amount with rawmaterials of cosmetics to be used as probiotics for cosmetics.

While the present disclosure has been described with reference to theparticular illustrative embodiments, it will be understood by thoseskilled in the art to which the present disclosure pertains that thepresent disclosure may be embodied in other specific forms withoutdeparting from the technical spirit or essential characteristics of thepresent disclosure. Therefore, the embodiments described above areconsidered to be illustrative in all respects and not restrictive.Furthermore, the scope of the present disclosure is defined by theappended claims rather than the detailed description, and it should beunderstood that all modifications or variations derived from themeanings and scope of the present disclosure and equivalents thereof areincluded in the scope of the appended claims.

1. A composition comprising: (a) a Lactobacillus plantarum CJLP475 strain deposited under Accession No. KCCM12287P; and (b) a Lactobacillus plantarum CJLP17 strain deposited under Accession No. KCCM12249P. 2.-3. (canceled)
 4. The composition of claim 1, wherein the composition has an antiviral activity against Porcine epidemic diarrhea virus (PEDV).
 5. The composition of claim 1, wherein the composition enhances immunity when administered to a subject.
 6. The composition of claim 5, wherein the subject is a livestock or pet.
 7. The composition of claim 1, wherein the composition increases antibodies in the body when administered to a subject.
 8. The composition of claim 1, wherein the composition, when administered to a subject, enhances immunity of the offspring born from the subject.
 9. The composition of claim 1, wherein the composition, when administered to a subject, alleviates the symptoms of a viral infection in the offspring born from the subject.
 10. The composition of claim 1, wherein (a) and (b) are in the form of a strain itself, a lysate thereof, a culture thereof, a concentrate thereof or a dried form thereof.
 11. The composition of claim 10, wherein the composition further comprises a cryoprotectant or an excipient.
 12. The composition of claim 11, wherein the cryoprotectant is at least one selected from the group consisting of glycerol, trehalose, maltodextrin, skim milk powder and starch, and the excipient is at least one selected from the group consisting of glucose, dextrin and skim milk.
 13. (canceled)
 14. A method for enhancing immunity of a subject, comprising: administering the composition according to claim 1 to a subject.
 15. A method for preventing or treating a virus-infected disease of a subject, comprising: administering the composition according to claim 1 to a subject.
 16. A method for preparing a probiotic composition, comprising: preparing each of a composition comprising a Lactobacillus plantarum CJLP475 strain deposited under Accession No. KCCM12287P; and a composition comprising a Lactobacillus plantarum CJLP17 strain deposited under Accession No. KCCM12249P; and mixing the composition comprising the CJLP475 strain and the composition comprising the CJLP17 strain.
 17. The method of claim 16, further comprising adding an additive to at least one of the composition comprising the CJLP475 strain, the composition comprising the CJLP17 strain, or the probiotic composition.
 18. The method of claim 17, wherein the additive is a cryoprotectant, and the method further comprises a freeze-drying step after adding the additive.
 19. The method of claim 18, wherein the strain in the freeze-dried composition is in a live state.
 20. The method of claim 16, comprising preparing a predetermined amount of the composition into a package after the mixing step.
 21. The method of claim 20, wherein, in the packaging step, the composition is prepared into a package such that the total amount of strains including the CJLP475 strain and the CJLP17 strain is 10⁶ CFU/g or more. 